1. Field of the Invention
The present invention relates to gaining access to and extracting specific internal tissue or structure of seed, and in particular, to a more efficient method or system of doing so in a relatively high throughput manner. The invention also relates to collecting extracted tissue or structure, including for evaluation by chemical, physical, or genetic testing, and in one aspect high throughput extraction of embryos suitable for enabling doubled haploid plant reproduction. One example of internal tissue or structure is the embryo of a maize seed.
2. State of the Art
The nature of most seeds makes access to and/or extraction of relevant tissue or structure non-trivial. While this can be done, seed-by-seed, in a labor-intensive, manual way (e.g. using a knife and tweezers), there is a need for a method of doing so in a more time and resource efficient manner.
For example, one of the most labor intensive steps in the process of doubled haploid plant production, such as with maize, is the extraction of immature embryos from the developing kernels. For maize kernels the term “immature” is used to mean less than physiological maturity, which normally means less than approximately 30 days or so after pollination or prior to total solidification of the endosperm or after black layer formation.
Extraction of embryos is done by hand at present, often under sterile conditions, which is tedious and time-consuming. In the case of maize, the most relevant genetic material is in the embryo of the seed. The embryo is encased by the endosperm. The endosperm, in turn, is encased in the pericarp, a relatively robust tissue. Additionally, maize kernels are relatively small in size, with the embryo being even smaller. Furthermore, the maize embryos and kernels develop biologically and temporally on a cob into an ear of corn, with a single ear generally having anywhere from a few to a few hundred kernels depending on genotype and growth conditions during plant and ear development. This increases the difficulty of extracting intact embryos. This also increases the complexity and difficulty of trying to automate, at least partially, the steps for accessing and then extracting an intact viable embryo, or a part of its tissue without destroying it or making it difficult to obtain accurate information from, or so that it is suitable for doubled haploid plant production.
There is also other internal tissue or structure in maize seed that may be of interest for efficient extraction. There are also other seed types and species which have at least some of the same or similar issues to maize and which have internal tissues or structures that could be of interest to access by non-destructive extraction.
Manual embryo extraction, especially on a seed-by-seed or kernel-by-kernel basis, is not a high throughput process. In commercial product development operations, time can be of the essence. Hundreds, if not thousands, of seed samples may need to have embryo tissue extracted in a relatively short time. With a manual process, substantial labor resources must be allocated to the task in order to achieve extraction of large numbers of embryos or other seed tissues in this short time frame, for subsequent analysis or use. Consider the example of a substantial size maize seed company. It may handle hundreds of thousands, and more often millions, of seeds per year. Depending on the age, attained growth and stage of development of the embryos, it is possible that present state of the art manual extraction methods could result in embryo extraction rates as low as 600 embryos per worker per hour, and in the best case scenario as much as 800 embryos per worker per hour. At this rate, as an example, processing 1 million seed would take well over a thousand worker hours. Moreover, embryo extraction rates using manual extraction techniques can vary as much as 300-400 embryos per worker per hour depending on the stage of development of the embryos (i.e., the number of days that have elapsed since pollination).
In commercial maize seed research and plant production, knowledge of a seed's phenotype and genotype saves time and effort by avoiding the need to grow plants from the seed and subsequently test plant tissue to look for desirable heritable traits or characteristics, thereby justifying its use in subsequent research and/or commercialization. Notwithstanding this effort, growing experimental plots of plants from seed for selection for commercial quantities is widely practiced in the maize seed business. Eliminating time and resources needed for this step is beneficial in a number of ways. Both time and significant resources in terms of labor and processing (e.g. planting, tilling, maintenance, obtaining tissue samples, etc.) would be saved. Moreover, the need for substantial amounts of land for growing the requisite plots could be greatly reduced. Decisions about a plant could be made when seed is developing or yet immature, as opposed to waiting for one or more subsequent generations to be grown. Additionally, efficient extraction of viable embryos could be used to harvest embryos for further use such as in the reproduction of succeeding generations of plants, and is particularly suitable for use in the process of doubled haploid plant production. This would facilitate shorter time to market, use of less space, time and resources, and the harvest of more embryos, more quickly.
Similar issues to those set forth above exist for other plants and seeds. It is beneficial to be able to obtain reliable information about a seed to make decisions about that plant without having to grow a subsequent generation and test tissue from the plant to make such decisions. It is also beneficial to extract, in a relatively high throughput manner, internal tissue(s) or structure(s) of the seed. It is therefore beneficial to quickly and efficiently extract internal tissue or structure from maize or other species.
Thus, a need exits for a more efficient way of harvesting or extracting embryos or embryo tissue from maize kernels for use in doubled haploid plant production. In this context, “efficiency” means a higher throughput of extracting embryos or tissues from multiple kernels in a given time period. Efficiency can also mean extraction of the embryo tissue without destroying it (i.e., extraction of intact viable embryos) or requiring substantial post-extraction steps to isolate the embryo tissue. Analogous issues and needs exist with other internal tissue or structure of maize and with internal tissue or structure of other types of seed plants. Benefits from an efficient and accurate method of accessing and extracting relevant internal seed tissue or structure exist for other such seed, as well as maize seed.